We plan to achieve a better understanding of the molecular basis of plasminogen activation by urokinase (UK) by studying the primary structure, particularly its active center and other reactive sites. This structural work will utilize the highly purified UK forms we have isolated, known UK inhibitors such as DFP and a newly synthesized fluorescent peptide chloroketone which we have found to react with the active-site histidine and can be used as a fluorescent probe. Peptide identification and isolation will be carried out by procedures such as gel electrophoresis, scintillation autoradiography, fluorescence spectrophotometry and affinity chromatography. The Edman procedure, including the ultra-micro solid-phase technique, will be used for amino acid sequencing. We will assess the relative thrombolytic potential of the two UK forms by measuring their affinities for plasmin(ogen) and fibrin(ogen) insolubilized on Sepharose. We also plan to determine the physiologic form(s) of the activator and identify the reactive group(s) of the enzyme involved in activating plasminogen;; we hope this will lead to optimal therapeutic use of the most effective UK form and eventually to the design of synthetic agents for plasminogen activation in vivo.